BASIC RESEARCH www.jasn.org Visualization of Calcium Dynamics in Kidney Proximal Tubules † † Kornélia Szebényi,* András Füredi,* Orsolya Kolacsek,* Rózsa Csohány, Ágnes Prókai, ‡ | Katalin Kis-Petik, Attila Szabó,§ Zsuzsanna Bősze, Balázs Bender,¶ József Tóvári,** †† ‡ Ágnes Enyedi, Tamás I. Orbán,* Ágota Apáti,* and Balázs Sarkadi* *Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary; †First Department of Pediatrics, ‡Department of Biophysics and Radiation Biology, MTA-SE Molecular Biophysics Research Group, and ††Second Department of Pathology, Semmelweis University, Budapest, Hungary; §MTA-SE, Pediatrics and Nephrology Research Group, Budapest, Hungary; |NARIC-ABI, Gödöllö, Hungary; ¶ImmunoGenes Kft., Budakeszi, Hungary; and **Department of Experimental Pharmacology, National Institute of Oncology, Budapest, Hungary ABSTRACT Intrarenal changes in cytoplasmic calcium levels have a key role in determining pathologic and pharmacologic responses in major kidney diseases. However, cell-specific delivery of calcium-sensitive probes in vivo remains problematic. We generated a transgenic rat stably expressing the green fluorescent protein-calmodulin– based genetically encoded calcium indicator (GCaMP2) predominantly in the kidney proximal tubules. The transposon-based method used allowed the generation of homozygous transgenic rats containing one copy of the transgene per allele with a defined insertion pattern, without genetic or phenotypic alterations. We applied in vitro confocal and in vivo two-photon microscopy to examine basal calcium levels and ligand- and drug-induced alterations in these levels in proximal tubular epithelial cells. Notably, renal ischemia induced a transient increase in cellular calcium, and reperfusion resulted in a secondary calcium load, which was signif- icantly decreased by systemic administration of specific blockers of the angiotensin receptor and the Na-Ca exchanger. The parallel examination of in vivo cellular calcium dynamics and renal circulation by fluorescent probes opens new possibilities for physiologic and pharmacologic investigations. J Am Soc Nephrol 26: 2731–2740, 2015. doi: 10.1681/ASN.2014070705 The mammalian kidney is a highly complex organ, problem may be avoided by a targeted, stable expres- and physiologic and pharmacologic modulation of sion of fluorescent sensor protein chimeras, enabling intrarenal calcium homeostasis has hardly been quantitative evaluation of cellular calcium dynamics. explored yet.1–3 Proximal tubular epithelial (PTE) Recent developments in two-photon microscopy, al- cells are among the cell types most sensitive to ische- lowing analysis of rapid changes of different fluores- mia and nephrotoxicity. Cellular calcium changes are cence intensities relatively deep in living tissues, key players in these responses; thus, exploration of cellular mechanisms underlying the pathophysiology of kidney injuries would have an utmost importance Received July 24, 2014. Accepted January 8, 2015. in drug development and the treatment of kidney Á.A. and B.S. contributed equally to this work. diseases. Calcium-dependent mechanisms in ische- Published online ahead of print. Publication date available at mia-reperfusion lead in many cases to irreversible www.jasn.org. cellular damage.4,5 Therefore, a better understanding of these mechanisms is essential to improve outcome Correspondence: Dr. Ágota Apáti or Dr. Balázs Sarkadi, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian for kidney transplantations. Academy of Sciences, Magyar Tudósok Körútja 2, Budapest, In vivo cellular calcium dynamics studies are 1117, Hungary. Email: [email protected] or sarkadi@bio- greatly hindered by the difficulty of delivering membrane.hu calcium-sensitive probes to specificcells.This Copyright © 2015 by the American Society of Nephrology J Am Soc Nephrol 26: 2731–2740, 2015 ISSN : 1046-6673/2611-2731 2731 BASIC RESEARCH www.jasn.org opened the possibility of performing in vivo studies for calcium immunostained kidney sections (Figure 1, B and C, Supplemen- dynamics in the mammalian kidney.6–8 Preferential use of rats in tal Figure 1, A and B), expression of both GCaMP2 and GGT1 pharmacokinetic and pharmacodynamic studies emphasizes the was much lower in the glomeruli, the distal and collecting tu- importance of calcium monitoring in tissues of this model animal. bules, or the vascular tissues. To explore renal calcium dynamics, we have generated transgenic In immunostaining of isolated cultures of PTE cells, the (TG) rats stably expressing a calcium indicator protein predomi- GCaMP2expressioncolocalizedwithmarkersofthePTepithelia, nantly in kidney proximal tubules (PTs), and we used fluorescence documented here by GGT1 costaining (Figure 1, D–F). PTE cells microscopy to study changes in renal intracellular calcium levels. stained positive for ATP binding cassette transporter G2, a PTE For efficient insertion of the transgene reporter construct marker (Supplemental Figure 1, D and E). into rat zygotes, we have used the hyperactive Sleeping Beauty Inthefollowingexperimentsfluorescenceintensitychangesof transposon. This approach promoted the generation of TG rat GCaMP2 were characterized in cultured PTE cells. As shown in founders at high frequencies9 and allowed selection of homo- Figure 2 and Supplemental Figure 2, addition of ATP, angioten- zygous TG rats, which contained one copy of the transgene/ sin II (AngII), trypsin, adrenaline, or histamine significantly haploid genome with a defined insertion pattern, with no ma- increased cellular free calcium levels. At the end of each exper- jor genetic or phenotypic alterations (see Concise Methods). iment we calibrated the calcium response by 5 mM ionomycin, For the intracellular fluorescent calcium indicator, we producing maximum cellular calcium load, followed by the ad- applied the genetically encoded calcium indicator (GCaMP2) dition of excess EGTA, decreasing intracellular calcium to the construct, a genetically engineered calmodulin-based calcium minimum. We found that calcium release from PTE cells in sensor fused to a green fluorescent protein (GFP)–based fluo- calcium-free medium by the addition of the sarcoplasmic/ rescent protein.10 Changes in light intensity emitted by this endoplasmic reticulum Ca(2+)–dependent ATPase pump in- fluorescent protein can be directly used to determine changes hibitor thapsigargin+ATP induced a major calcium-induced in intracellular free calcium concentration. The GCaMP2 pro- calcium influx (Supplementary Figure 2D). tein has already been applied in various in vivo measurements As shown in Figure 2, A and B, addition of ATP (5–100 mM) within selected tissue preparations and in transgenic mice.11–14 produced a large increase in intracellular free calcium in PTE Although new versions of genetically engineered calcium indi- cells. A second ATP addition had no such effect (Supplemental cators are also available, the GCaMP2 construct was reliably and Figure 2E), and pretreatment of the cells with suramin (200–600 efficiently working in our hands in various mammalian cellular mM), a P2 purinergic receptor inhibitor, eliminated ATP- systems. As reported,15 in human stem cells calcium imaging induced calcium signal (Figure 2, A and B). These findings indicate could be performed without the need for potentially toxic dye calcium signaling through P2 purinergic receptors in PTE cells. loading. We found that AngII (10 mM) induced a measurable calcium In TG rats, the GCaMP2 calcium indicator expression was signal in isolated PTE cells, although the amplitude of calcium driven by a CAG promoter,16 which we previously applied for transients detected in individual cells showed high diversity (Fig- generating a calcium reporter system in human embryonic ure 2C). Repeated AngII addition did not cause repeated calcium stem cells.15 In TG rats we found especially high GCaMP2 effects, and the specific inhibitor of angiotensin receptor type 1 expression in renal PTE cells. For in vitro imaging we applied (AT1), active form of candesartan (1 mM),17 fully blocked confocal microscopy, while for in vivo studies we used two- AngII-induced calcium response (not shown). photon microscopy with multicolor detection and rapid re- In various kidney diseases and in the process of kidney sponse software capabilities (see Concise Methods). transplantation, ischemia and reperfusion cause substantial damage in the PTs, especially by changing calcium homeostasis.3 In an attempt to mimic ischemia (hypoxia) and reperfusion RESULTS (reoxygenation) in isolated renal PTE cells, we have pretreated the cells in media containing 200 mMCoCl2, reported to cause GCaMP2 Expression in Kidney PT Cells: In Vitro hypoxic conditions in various cell types in vitro.18,19 After 24 Confocal Microscopy Experiments hours’ pretreatment, exchange of the media for oxygenated me- Kidneys of rats stably expressing the GCaMP2 indicator protein dia containing no CoCl2 resulted in a large transient increase in were examined by preparing frozen tissue slices and by establish- cellular calcium levels, while addition of ATP still produced a ing long-term cultures of dissociated cell types. In these prepa- rapid calcium rise (Figure 2D). The exchange of media without rations we examined GCaMP2 expression by anti-GFP antibody CoCl2 pretreatment did not alter intracellular calcium levels staining to bypass calcium-level dependent heterogeneity of the (Supplemental Figure 2F). GCaMP2 signal. Because data in the literature